An oomycete NLP cytolysin forms transient small pores in lipid membranes

Microbial plant pathogens secrete a range of effector proteins that damage host plants and consequently constrain global food production. Necrosis and ethylene-inducing peptide 1–like proteins (NLPs) are produced by numerous phytopathogenic microbes that cause important crop diseases. Many NLPs are cytolytic, causing cell death and tissue necrosis by disrupting the plant plasma membrane. Here, we reveal the unique molecular mechanism underlying the membrane damage induced by the cytotoxic model NLP. This membrane disruption is a multistep process that includes electrostatic-driven, plant-specific lipid recognition, shallow membrane binding, protein aggregation, and transient pore formation. The NLP-induced damage is not caused by membrane reorganization or large-scale defects but by small membrane ruptures. This distinct mechanism of lipid membrane disruption is highly adapted to effectively damage plant cells.


Supplementary Text S1. Modeling of the fluorescent signals in the microfluidic experiments
Microfluidic experiments yield data for the membrane-bound fluorescently labeled NLP protein (NLPPya-A488), cNLP (t), and the concentration of the fluorescent dye A594 in the giant unilamellar vesicle (GUV) interior, cA594 (t). Here we use these data to test two possible models of NLPPya-induced membrane leakage from a GUV.
The A594 leakage depends on the following variables and parameters: concentration of membrane-bound NLPPya (this is a measured parameter, corresponding to the intensity of A488, here expressed in dimensionless arbitrary units) cA594 (t) concentration of A594 in the vesicle (this is a measured parameter, normalized to the maximal intensity of the A594 signal, here expressed in dimensionless arbitrary units) cmono (t) concentration of NLPPya monomers on the membrane (dimensionless units) ccl (t) concentration of clustered NLPPya on the membrane (dimensionless units) P (t) membrane permeability (in (m/s)) R GUV radius (in (m)) A GUV surface area (A = 4πR 2 ) V GUV volume (V = 4πR 3 / 3)

a) Leakage of A594 from the vesicle
Assuming that cA594 inside the vesicle is homogeneous (i.e., that diffusion of A594 within the vesicle is much faster than the leakage of A594 out of the vesicle), cA594 (t) can be described by a standard equation for solute leakage:

Simple model assuming that membrane permeability is proportional to the membrane-bound NLPPya
Within this model, the membrane permeability can be written as , where kp is a membrane permeability coefficient (in (m/s)). In this case, the concentration of A594 inside the vesicle follows a simple kinetic equation: , where Kp is the only kinetic constant for A594 leakage (it is related to the size of the vesicle and the permeability coefficient, Kp = 3 kp / R, in (s -1 )). Within this model, the A594 starts leaking as soon as the protein concentration on the membrane increases (the value of Kp only affects the slope of the leakage curve, i.e. the leaking rate) and thus the model cannot account for the observed time-lag ( fig. S15A). In addition, the time-lag cannot be reproduced even if one assumes that membrane permeability depends on cooperative action of NLPPya proteins on the membrane, i.e., if , where is the number of participating NLPPya monomers in an induced pore.

A minimal two-step model of slow nucleation and fast auto-catalytic growth
We continue with the assumption that NLPPya clusters have to form in order for the membrane to become permeable. Assuming that membrane permeability P(t) is proportional to the amount of NLPPya clusters in the membrane , the concentration of A594 can be calculated from: .
We then assume that NLPPya clusters form from NLPPya monomers through a 2-step Finke-Watzky minimalistic model of nucleation and growth (26). According to this model, the concentration of clusters follows: , where k1 and k2 correspond to nucleation and assembly growth rates in (s -1 ), respectively (25). In this case, the sum of the amounts of clusters and monomers equals the total amount of the membrane-bound NLPPya, which is proportional to the measured fluorescence signal: .
As presented in fig. S15B, this model readily describes both the time-lag and the rate of the A594 leakage.     The hydrogen bonding network at the membrane interface. The scheme displays the following: POPC lipids (orange carbon atoms), GIPC lipids (green carbon atoms), phosphate groups (van der Waals spheres), NLPPya (magenta), the residues establishing hydrogen-bonding interactions with the membrane (magenta carbon atoms), oxygen (red), hydrogen (white), nitrogen, the tryptophan 155 (W155; presented as van der Waals spheres) and the C-terminal helix (blue), Mg 2+ (gold van der Waals spheres). The three insets show a magnified view of the NLPPya interactions with the membrane and at the C-terminal helix (left panel) and at the binding cavity (right top and bottom panel). NLPPya binding is mostly stabilized by electrostatic interactions (i.e. the electrostatic and van der Waals components of total interaction energy between NLPPya and the membrane are of -240.9 ± 5.7 and -57.4 ± 4.5 kcal/mol, respectively).    The following is displayed: the substrate layer distributions (silicon (black) and silicon dioxide (gray)), lipid head groups (green), lipid tails (red), and water distribution across the membrane (blue). Line widths depict the ambiguity in component position and volume fraction as 65% confidence intervals of the acceptable parameter ranges determined from Monte Carlo resampling of the experimental data fits in (A).

Fig. S10. Neutron reflectivity data and scattering length density (SLD) profiles for a POPC:sterols 7:3 supported lipid bilayer after h-NLP Pya binding.
Reflectometry data for each isotopic contrast (left) and the SLD profiles (right). The shaded areas depict the 65% confidence intervals of the model parameters determined from Monte Carlo resampling of the experimental data fits. Gray profiles were obtained from the neutron reflectivity data from the silicon surface prior to the deposition of the supported lipid bilayer.      The protein also establishes stable interactions with several other GIPC's head groups. The initial interaction with the plant membrane is predominantly electrostatically driven. NLPPya further assembles into aggregates that are heterogeneous in size and shape (iii). GIPC's very long fatty acids can have a structural role in coupling both membrane leaflets (14). Transient membrane ruptures occur after a membrane is saturated with NLPPya, which cause the redistribution of GIPCs due to numerous contacts with NLPPya. In this model, NLPPya resides on the membrane surface and does not penetrate deeply into the bilayer throughout the pore formation process. According to MD simulations, the regions that may be responsible for membrane rupture formation are the loops around the GIPC binding site and the C-terminal helix. Legend: NLPPya (magenta), sterols (gold), GIPC sugar head group (green), and GIPC inositol phosphorylceramide part (white). Movie S1. A time-lapse recording of the binding of 300 nM NLPPya to a DOPC:GIPC:sterols 1:1:1 supported lipid bilayer in 20 mM MES, pH 5.8. Scan area: 150 nm × 150 nm; scan rate: 0.5 frame/s.

Movie S4.
A time-lapse recording of a representative experiment showing 12 giant unilamellar vesicles filled with A594 and exposed to NLPPya-A488 in a microfluidic diffusion chamber. The movie shows the bright field image of the chamber (top) along with the red fluorescent channel for A594 (middle) and the green fluorescent channel for A488 (bottom). At 200 s, an iso-osmolar NLPPya-A488 glucose solution is introduced into the main channel and begins to diffuse into the chamber from the main channel on the left. At approximately 600 s, NLPPya-A488 reaches the vesicles and begins to bind to the membranes. The side of the membrane oriented towards the entrance of the chamber exhibits a more intense green signal compared to the opposite side of the vesicle. At approximately 850 s, the first vesicle starts to leak, as deduced from a diminishing red signal from the interior of the vesicle. The other vesicles then follow one-by-one. At 2150 s, the NLPPya-A488 solution is washed away from the main channel and subsequently also from the diffusion chamber. The NLPPya-A488 signal remains on the membrane of the vesicles.